Journal of Experimental Botany, Vol. 51, No. 346, pp. 901-910,
May 2000
© 2000 Oxford University Press
Does ascorbate in the mesophyll cell walls form the first line of defence against ozone? Testing the concept using broad bean (Vicia faba L.)
1 Air Pollution Laboratory, Department of Agricultural and Environmental Science, Ridley Building, The University, Newcastle Upon Tyne NE1 7RU, UK
2 Biological Research Centre, Hungarian Academy of Sciences, Institute of Plant Biology, H-6701 Szeged, Temesvári krt. 62, PO Box 521, Hungary
3 Institute of Agricultural Engineering—Bornim (ATB), Max-Eyth-Allee 100, D-14469 Potsdam-Bornim, Germany
Received 16 July 1999; Accepted 3 January 2000
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Abstract |
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Broad bean (Vicia faba L.) plants were exposed, in duplicate controlled environment chambers, to charcoal/Purafil®-filtered air (CFA-grown plants) or to 75 nmol mol-1 ozone (O3) for 7 h d-1 (O3-grown plants) for 28 d, and then exposed to 150 nmol mol-1 O3 for 8 h. The concentration of ascorbate (ASC) was determined in leaf extracellular washing fluid (apoplast) and in the residual leaf tissue (symplast) after 0, 4 and 8 h acute fumigation, and after a 16 h ‘recovery’ period in CFA. Changes in stomatal conductance were measured in vivo in order to model pollutant uptake, while the light-saturated rate of CO2 assimilation (Asat) was recorded as an indicator of O3-induced intracellular damage. Measurements of Asat revealed enhanced tolerance to 150 nmol mol-1 O3 in plants pre-exposed to the pollutant compared with equivalent plants grown in CFA, consistent with the observed reduction in pollutant uptake due to lower stomatal conductance. The concentration of ASC in the leaf apoplast (ASCapo) declined upon O3-treatment in both CFA- and O3-grown plants, consistent with the oxidation of ASCapo under O3-stress. Furthermore, the decline in ASCapo was reversible in O3-grown plants after a 16 h ‘recovery’ period, but not in plants grown in CFA. No significant change in the level and/or redox state of ASC in the symplast (ASCsymp) was observed in plants exposed to 150 nmol mol-1 O3, and there was no difference in the constitutive level of ASCsymp between CFA- and O3-grown plants. Model calculations indicated that the reaction of O3 with ASCapo in the leaves of Vicia faba is potentially sufficient to intercept a substantial proportion (30–40%) of the O3entering the plant under environmentally-relevant conditions. The potential role of apoplastic ASC in mediating the tolerance of leaves to O3 is discussed.
Key words: Apoplast, cell wall, ozone, detoxification, antioxidants, ascorbate.
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Introduction |
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Tropospheric concentrations of ozone (O3) are known to pose a growing threat to the vitality of natural and managed ecosystems in many parts of the industrialized world (Runeckles and Chevone, 1992
; Davison and Barnes, 1992, 1998; UNECE, 1996; Fuhrer et al., 1997). However, the mechanisms underlying the phytotoxicity of this ubiquitous air pollutant are just beginning to be unravelled (Harris and Bailey-Serres, 1993; Kangasjärvi et al., 1994; Mudd, 1996; Sharma and Davis, 1997). One area of growing interest has been the role played by ASC, and other potential antioxidants situated in the leaf apoplast, in mediating the tolerance of plant tissues to this powerful oxidant (Polle and Rennenberg, 1993; Lyons et al., 1999a).
The flux of O3 to the leaf interior is controlled almost entirely by stomatal conductance (Kerstiens and Lendzian, 1989). Once inside the leaf, the pollutant dissolves into the aqueous matrix which overlays the surface of the cells lining the sub-stomatal cavity (the apoplast), resulting in an intercellular space O3 concentration that is demonstrably close to zero (Laisk et al., 1989). Ozone is a strong oxidant (+2.07 V), and is considered to react first with oxidizable constituents of the apoplast, and, subsequently, if the pollutant and/or its reactive products escape interception, with components of the plasmalemma and cytosol (Heath, 1980, 1988). The dissolution chemistry of O3 in the region of the cell wall is far from fully understood (Heath, 1980, 1988; Mudd, 1996; Moldau, 1998). However, the primary reactions of the pollutant have been suggested to yield several other potentially damaging reactive oxygen species (ROS), including the hydroxyl radical, superoxide, singlet oxygen, and hydrogen peroxide (Grimes et al., 1983; Kanofsky and Sima, 1995a, b; Mehlhorn et al., 1990;Pryor, 1994; Byvoet et al., 1995), and possibly organic peroxides and hydroperoxides through the reaction of O3 with biogenic hydrocarbons (Salter and Hewitt, 1992). Consequently, two main factors are considered to govern the resistance of plants to O3: (i) the physical exclusion of the pollutant from sensitive intracellular targets (O'Dell et al., 1977; Runeckles, 1992; Barnes et al., 1999a) and (ii) the capacity to intercept and detoxify O3, and/or its reactive products, ideally before reaction with components of the plasmalemma and cytosol (Heath, 1988; Chameides, 1989; Polle and Rennenberg, 1993; Moldau, 1998; Lyons et al., 1999a).
Over the past decade, growing attention has been paid to the role played by certain constituents of the leaf apoplast in attenuating the flux of O3 impinging on the plasmalemma, the primary site of O3 damage. The aqueous matrix of the cell wall is known to contain many compounds which can act as antioxidants, including ascorbate (ASC) and dehydroascorbate (DHA) (Castillo and Greppin, 1988; Polle et al., 1990; Takahama and Oniki, 1992; Takahama, 1993; Luwe et al., 1993; Luwe and Heber, 1995; Deutsch 1998a, b), polyamines and phenolics (Langebartels et al., 1991; Eckey-Kaltenbach et al., 1993, 1994), glutathione (Polle et al., 1990; Jamaï et al., 1996), as well as several enzymes which are well known to protect against ROS; Cu/Zn superoxide dismutase (Streller and Wingsle, 1994; Ogawa et al., 1996; Lyons et al., 1999b), glutathione-S-transferase (Flury et al., 1996) and peroxidases (Castillo et al., 1984, 1987; Takahama and Oniki, 1992; Polle et al., 1994; Castillo and Greppin, 1986; Peters et al., 1989; Ranieri et al., 1996). Recent studies (Vanacker et al., 1998a, b) suggest that catalase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase may also be present in the apoplast of some species, but these findings require confirmation.
In terms of potential detoxification capacity, attention has focused principally on the reaction of O3 with ASC (Chameides, 1989; Polle and Rennenberg, 1993), this compound tops the hierarchical series of cell wall reaction-targets, based on the results of ex vivo (Kanofsky and Sima, 1995b) and in vitro (Giamalva et al., 1985; Kanofsky and Sima, 1995a; Mudway and Kelly, 1998) studies. The aim of the present investigation was to use broad bean (Vicia faba L.) as a convenient model to examine the impacts of exposure to environmentally-relevant O3 concentrations on ASC/DHA levels in the leaf apoplast/symplast in vivo, to probe the relationship between ASC/DHA status and O3 tolerance, and to estimate the theoretical extent of O3 protection afforded by the reaction of O3 with apoplastic ASC.
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Materials and methods |
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Plant culture and fumigation
Seed of broad bean (Vicia faba L. cv. Imperial White Windsor) was germinated in seed trays containing vermiculite, in duplicate controlled environment chambers ventilated with either charcoal/Purafil®-filtered air (CFA <5 nmol mol-1 O3) or CFA plus 75 nmol mol-1 O3 for 7 h d-1 (O3; 10.00 h–17.00 h) at a flow rate sufficient to achieve
2 air changes min-1 in each chamber. Air temperature was controlled on a dynamic basis from a maximum of 24±1 °C during the day, to a minimum of 16±1 °C at night. Each chamber was illuminated by two metal-halide floodlights (Siemens 400 W luminaires fitted with HQI/TS 400 floodlights) providing a photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 at plant height, supplied as a 14 h photoperiod (07.00 h–21.00 h). Relative humidity was maintained at 65±5%. All plants were transplanted into larger pots (3 dm3) containing a standard potting compost (John Innes No. 2) after 7 d fumigation. Further details of this controlled environment fumigation facility, and gas control/monitoring systems are provided elsewhere (Barnes et al., 1995; Zheng et al., 1998). Experiments were performed on the fourth fully expanded leaf on 4-week-old plants. Acute O3 exposures (150 nmol mol-1, 8 h) were administered in a controlled environment chamber identical to those in which plants were grown, ventilated by the same air handling system. Plants were returned to their respective growth conditions following O3-treatment.
Growth and dry matter partitioning
Between 10 and 12 plants (5–6 plants per chamber) grown in CFA and O3 were harvested after 28 d. An initial harvest of 10 plants (5 plants per chamber) was made after 7 d—the root separated from the shoot, component plant parts dried (70 °C for 1 week) and then weighed. Mean plant relative growth rate (R), the relative growth rate of root (RR) and shoot (RS), and allometric root/shoot growth (K; RR/RS) were calculated as described previously (Hunt, 1990).
Leaf gas exchange measurements
In situ measurements of leaf gas exchange were made on the fourth (youngest fully expanded) leaf borne on six ‘control’ CFA- or O3-grown plants and an equivalent number of plants transferred into 150 nmol mol-1 O3, at regular intervals over the course of the day. Additional measurements were made on the same leaves following 16 h recovery under pretreatment conditions. Experiments were repeated twice; the data presented representing the average of both experiments (i.e. n=12). Rates of CO2/H2O exchange were monitored using a portable infra-red gas analysis system (CIRAS-1 portable IRGA system, PP Systems, Hitchin, Herts, UK). Measurements were made at a cuvette CO2 concentration of 350±1 µmol mol-1 under chamber conditions (PPFD=198±4 µmol m-2 s-1 at the position occupied by the leaf in the cuvette; leaf temperature=24±0.5 °C) using a standard Parkinson leaf cuvette (model PLC-B, PP Systems), and under light-saturated conditions (PPFD=1200±2 µmol m-2 s-1 at the position occupied by the leaf; leaf temperature=23±0.5 °C) using a Parkinson leaf cuvette that incorporates automated light and temperature control (model auto-PLC-B, PP Systems). The light-saturated rate of CO2 assimilation (Asat) and stomatal conductance to water vapour (gH2O; measured under chamber conditions) were calculated according to von Caemmerer and Farquhar (von Caemmerer and Farquhar, 1981).
Preparation of extracellular washing fluid and residual leaf extracts
Extracellular washing fluid (EWF) and residual leaf extracts (RLEs) were prepared from independent ‘control’ (CFA- or O3-grown) and ‘O3-treated’ (CFA- or O3-grown plants exposed to 150 nmol mol-1 O3) plants at regular intervals over the course of the fumigation. Additional measurements were performed on an equivalent number of plants allowed to ‘recover’ for a 16 h-period under pretreatment conditions. Experiments were repeated twice; the data presented represent the average across the two experiments (n=20–24 ‘control’; n=6 acute O3-treatment).
The fourth leaf (0.5 g FW) was detached, weighed, washed with double-distilled water, and then vacuum infiltrated (-70 kPa) with 100 ml of 66 mM K-phosphate buffer (pH 4.5) containing 100 mM KCl and 2.5 mM EDTA (two infiltration periods; each of 1 min duration). Immediately after infiltration, the leaf was blotted dry, re-weighed, rolled carefully and inserted into a syringe placed over a preweighed 1.5 ml Eppendorf tube containing 100 µl of cold 100 mM HCl. Extracellular washing fluid was collected under ‘soft’ centrifugation (5 min., 80 g) at 4 °C (3 K-18 centrifuge, Sigma-Aldrich, Poole, UK), and was kept on ice for only a short-time before the analysis of ASC/DHA. Eppendorf tubes were reweighed following centrifugation to determine the volume of recovered EWF. Approximately 500 µl of EWF was recovered per gram leaf fresh weight and there was no significant difference in the volume of EWF recovered (on the basis of leaf fresh weight) between plants grown in CFA and those exposed to O3. The maximum time between leaf detachment to the analysis of EWF was 10 min.
Residual leaf extracts (RLEs) were prepared by homogenizing the leaf tissue remaining after the isolation of EWF; 0.1 g FW of leaf tissue was ground in 2 ml of ice-cold 100 mM HCl containing 2.5 mM EDTA. The homogenate was transferred to 50 ml tubes and centrifuged at 10 000 g for 10 min at 4 °C. The supernatant was decanted and ASC/DHA content determined immediately.
Cytoplasmic contamination of EWF was checked by measuring the activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) employing the method of Kornberg and Horecker (Kornberg and Horecker, 1955) using the technique described in detail elsewhere (Lyons et al., 1999b). The activity of G6PDH in RLEs was 6.95±0.42 nkat g-1 FW, but G6PDH activity was below the limits of detection in EWF (detection limit 0.5 nkat g-1 FW); indicating that EWF was not contaminated with intracellular protein.
Ascorbate determination
All assays were performed at 25 °C using matched quartz cuvettes. Ascorbate/dehydroascorbate (ASC/DHA) content was determined using the spectrophotometric method described previously (Takahama and Oniki, 1992). For ASC, initial absorbance of a 50 µl aliquot of extract was measured at 265 nm in 100 mM K-phosphate buffer (pH 6.1), then remeasured following the addition of ascorbate oxidase (1 U ml-1). Complete oxidation of ASC took no longer than 1 min. Dehydroascorbate content was determined in another 50 µl aliquot. Initial absorbance was recorded as for ASC, and then the sample was remeasured following the addition of 2 mM DL-dithiothreitol (DTT). Complete reduction of DHA took no longer than 8 min. Measurements were corrected to account for the absorbance of DTT at 265 nm. An extinction coefficient of 14 mM-1cm-1 for ASC at 265 nm was used in calculations (Nakano and Asada, 1981). Recovery of internal ASC standards was 93±6% and 90±7% for EWF and RLEs, respectively, and there was no evidence of significant oxidation during the extraction process. The redox state of ASC was calculated as
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Modelling the extent of ozone protection afforded by apoplastic ascorbate
The fraction of O3 detoxified through the direct reaction with ASC in the mesophyll cell wall was estimated using a computer-based model described elsewhere (Plöchl et al., 2000). Measured input parameters are provided in Table 1. Cell wall thickness was determined from electron micrographs (according to Steer, 1981).
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Statistical analyses
Statistical analyses were performed using SPSS (SPSS Inc., Chicago, USA). Data were first subjected to analysis of variance (ANOVA) investigating the influence of chamber, growth conditions, O3-treatment, and time. No significant chamber effects were found within treatments, so data for individual plants were subjected to multivariate analysis of variance (MANOVA) to test the effects of growth conditions, O3-treatment and time under the assumption that plants in replicate chambers were as likely to be as similar, or as different from plants within an individual chamber. Significant differences were determined using the least significant difference (LSD) calculated at the 5% level. Independent t-tests were used to compare individual means. Correlations were performed using Prism (GraphPad Software, San Diego, California), employing least square linear regression methods to test the goodness of fit.
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Results |
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Visible injury
Chronic O3 exposure (28 d fumigation with CFA plus 75 nmol mol-1 O3 7 h d-1) resulted in no typical visible symptoms of injury on leaves at any stage of fumigation, nor were there any signs that O3 exposure accelerated the rate of leaf senescence.
Acute O3 exposure (150 nmol mol-1 8 h) resulted in the development of typical symptoms of visible damage (necrotic lesions developing on the adaxial leaf surface within 8 h of exposure, affecting 20% of the leaf area) on the older leaves of plants transferred from CFA. Equivalent exposure resulted in no visible symptoms on the leaves of O3-grown plants or on the leaf that was employed for all biochemical and physiological measurements (i.e. the youngest fully expanded leaf).
Growth and dry matter partitioning
Analysis of variance revealed no statistically significant effects of chronic O3 exposure on plant relative growth rate (RCFA=1.015±0.07; RO3=0·926±0.05) or allometric root:shoot growth (KCFA=0.765±0.06; KO3=0.717±0.04).
Leaf gas exchange
Figure 1 shows the effects of O3-treatment on the light-saturated rate of CO2 assimilation (Asat) and stomatal conductance (gH2O) for CFA- and O3-grown plants. Analysis of variance revealed that acute O3 exposure resulted in a highly significant (P<0.001) decline in Asat within 1 h in both CFA- and O3-grown plants; O3-treatment reduced Asat by 22% in CFA-grown plants and by 10% in O3-grown plants, the photosynthetic rate persisting at this reduced level for the remaining 8 h period of the treatment (Fig. 1A). Paired t-tests revealed a significant (P<0.05) depression in Asat following acute O3 exposure in CFA-grown, but not in O3-grown plants, while ANOVA revealed no statistically significant difference in the response of Asat to acute O3 exposure in plants with contrasting O3 histories. Acute O3 exposure was found to result in a parallel decline in Asat and gH2O (Fig. 1), but the average extent of the decline in gH2O with acute O3 exposure was significantly less in O3-grown plants (-21%) compared with their counterparts raised in CFA (-40%). Consequently, a strong O3-growth-concentrationxO3-treatment interaction (P<0001) was found. Differences in response were, at least partly, attributable to the fact that O3-grown plants exhibited lower (44%; P<0.001) gH2O at the start of the treatment compared with their counterparts raised in CFA.
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Effects of ozone on apoplastic ascorbate content
Figure 2 shows changes in the concentration and redox status of ASC in EWF following acute O3 exposure. Despite the considerable replication employed in an effort to minimize the variation commonly associated with such measurements (Luwe and Heber, 1995), MANOVA revealed the impacts of the acute O3-treatment on apoplastic ASC content (ASCapo) and redox state to be, more often than not, on the borderlines of statistical significance. Acute fumigation resulted in a 30% decline in ASCapo in both CFA- and O3-grown plants, within 4 h of exposure to the pollutant. Interestingly, no change in ASCapo was observed in CFA-grown plants after 16 h of ‘recovery’. However, ASCapo returned to pre-exposure levels within 16 h of transfer of O3-grown plants to ‘control’ conditions (P<0.05).
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After 4 h exposure to 150 nmol mol-1 O3, a 50% decrease in the concentration of DHA in the apoplast (DHAapo) of CFA-grown plants was observed (Fig. 2B
). In contrast, DHAapo in O3-grown plants declined by only 15% in response to the same treatment. Constitutive levels of DHAapo in CFA- and O3-grown plants were similar (Fig. 2B), and the redox state of ASC in the leaf apoplast of CFA- and O3-grown plants remained unchanged during 8 h fumigation with 150 nmol mol-1 O3. However, a higher (P<0.05) redox state was found in EWF isolated from O3-grown plants compared with their CFA-grown counterparts after 16 h ‘recovery’. The constitutive redox state of ASC in the leaf apoplast was similar in CFA- and O3-grown plants; 67±4% in CFA-grown plants and 74±3% in O3-grown plants (Fig. 2C).
Effects of ozone on symplastic ascorbate content
Exposure of CFA- and O3-grown plants to acute O3 resulted in no marked changes in ASC and DHA content in RLEs (Fig. 3). Symplastic ASC levels (ASCsymp) declined by 25% within 4 h of O3-treatment in the CFA-grown plants, but this effect was not statistically significant. Ozone-treatment resulted in no shift in the redox state of symplastic ASC in CFA- or O3-grown plants (Fig. 3C) and constitutive ASCsymp and DHAsymp contents were similar.
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A significant linear relationship (r2=0.44, P<0.0001) was found between corresponding measurements of ASCapo and ASCsymp for individual leaves (Fig. 4
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Modelling the extent of ozone protection afforded by apoplastic ascorbate
A linear relationship (r2=0.48, P<0.0001) was found between measured and modelled apoplastic ASC concentrations (Fig. 5).
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Modelled O3 fluxes are presented in Table 2
. Under chronic exposure (75 nmol mol-1 O3), the flux of O3 to the leaf interior (JINT; the flux of O3 determined purely by gH2O) and the flux of O3 impinging on the plasmalemma (JPLASMA; the flux of O3 determined by the combination of gH2O and the reaction of the pollutant with apoplastic ASC) were decreased (P<0.05) in O3-grown plants compared with theoretical maximum O3 fluxes at 75 nmol mol-1 (JMAX; the theoretical flux of O3 based on measurements of gH2O for CFA-grown plants assuming apoplastic ASC=0). The contribution of gH2O and apoplastic ASC to the observed decrease in JPLASMA in O3-grown plants was 0.66 and 0.38 nmol O3 m-2 s-1, respectively.
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Upon initial exposure to O3-treatment (150 nmol mol-1), JPLASMA was reduced (P<0.01) in both CFA- and O3-grown plants compared with the corresponding JMAX. JPLASMA was lower (P<0.01) in O3-grown plants than equivalent CFA-grown plants, predominantly as a result of a decrease (P<0.01) in JINT associated with shifts in gH2O rather than differences in ASCapo. The relative contribution of gH2O and ASCapo to the decline in JPLASMA in plants pre-exposed to O3 were 1.25 and 0.60 nmol O3 m-2 s-1, respectively. After 4 h of exposure to O3, there was no significant difference in JINT between CFA- and O3-grown plants due to a decline (P<0.01) in JINT in the CFA-grown plants. The predicted detoxification of O3 by ASCapo was not significantly different between plants raised in CFA and O3, but JPLASMA was lower (P<0.001). The relative contribution of gH2O and ASCapo to decreases in JPLASMA after 4 h exposure to 150 nmol mol-1 O3 were 1.16 and 0.51 nmol O3 m-2 s-1 (CFA-grown) and 1.60 and 0.63 nmol O3 m-2 s-1 (O3-grown), respectively.
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Discussion |
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The concentration of ASC in the apoplast of fully-expanded Vicia faba leaves was 0.50±0.01 mM in CFA- and 0.59±0.01 mM in O3-grown plants, and 70±4% of the apoplastic ASC pool was in the reduced state, irrespective of whether plants were raised in CFA or at environmentally-relevant O3 concentrations. These findings agree well with data on ASCapo for unstressed leaves of another cultivar of Vicia faba (Luwe and Heber, 1995
), as well as with data for the leaves of other species (Sedum album L.: Castillo and Greppin, 1988; Spinacia oleracea L., Takahama and Oniki, 1992; Luwe et al., 1993; Nicotiana tabacum L.: Batini et al., 1995; Cucurbita pepo L.: Ranieri et al., 1996; Kalanchoë daigremontiana Hamet et Perr.: Takahama, 1993; Phaseolus vulgaris L.: Moldau et al., 1997, 1998).
Exposure of CFA- and O3-grown plants to 150 nmol mol-1 O3 was found to cause a decline in the ASC content of the leaf apoplast in vivo (Fig. 2). These findings suggest that ASCapo is consumed upon exposure to O3—a conclusion supported by a wealth of in vitro studies which have demonstrated O3-driven oxidation of ASC in pure biochemical solutions (Giamalva et al., 1985; Mudway and Kelly, 1998), EWF (Kanofsky and Sima, 1995b), respiratory tract lining fluid (Kelly et al., 1995), and blood plasma (Cross et al., 1992; van der Vliet et al., 1995). Furthermore, the findings in the present study are consistent with O3-induced changes in the composition of the leaf apoplast observed in several species (Castillo and Greppin, 1988; Luwe et al., 1993; Polle et al., 1995; Luwe and Heber, 1995), but are not consistent with the observations made by Jakob and Heber (Jakob and Heber, 1998). After 4 h exposure to 150 nmol mol-1 O3, the decline in ASCapo was found to attain a steady state (Fig. 2). This finding suggests the attainment of an equilibrium between the rate of O3-induced oxidation of ASCapo and the replenishment of the ASC pool from the symplast. The concentration of ASC in the leaf apoplast is believed to be determined by a combination of free diffusion of the neutral species (ascorbic acid) (Plöchl et al., 2000; Bichele et al., 2000) and the facilitated diffusion of ASC across the plasmalemma (Foyer and Lelandais, 1996; Horemans et al., 1996), a view consistent with the linear relationship observed between corresponding measurements of ASCapo and ASCsymp on individual leaves (Fig. 4). Following a period of ‘recovery’ in CFA, the O3-induced decline in ASCapo was found to be reversible in plants pre-exposed to the pollutant, but not in those grown in CFA. A possible explanation for this is that the oxidation of ASC in the apoplast continued in CFA-grown plants after the cessation of fumigation due to the elicitation of an unidentified source of oxidative stress (Schraudner et al., 1998; Rao and Davis, 1999).
The oxidized product of the ASC : O3 reaction, DHA, cannot be reduced efficiently in the apoplast and must return to the cytosol for recycling (Castillo and Greppin, 1988; Polle et al., 1990; Luwe et al., 1993). The results of the present study indicated that ASC consumption in the apoplast was not mirrored by an increase in the concentration of DHA and as a consequence no shifts in the redox state of ASCapo were observed (Fig. 2C). Similar findings were reported by Polle and co-workers (Polle et al., 1995) during their studies on the effects of O3 on apoplast/symplast antioxidant status in Picea abies [L.] Karst. These findings imply that there is rapid import of DHA into the cytosol under O3 fumigation, a contention supported by the work of Horemans et al. which suggests that the carrier-mediated system for ASC/DHA on the plasmalemma has a strong preference for DHA, and/or DHA breaks down and is irreversibly lost from the apoplast (Horemans et al., 1997, 1998). Recent studies by Deutsch indicate that DHA itself may be a powerful antioxidant (Deutsch, 1998a, b). Hence, it is possible that the ‘loss’ of DHA from the apoplast may be the result of reaction with (and detoxification of) O3. Further studies are required to confirm this.
In the present study, the increase in tolerance of O3-grown plants in comparison with their CFA-grown counterparts appeared not to be mediated by differences in the potential detoxification of the pollutant by ASCapo, but by the enhanced exclusion of O3 through a decline in stomatal conductance in plants pre-exposed to the pollutant. However, model calculations revealed that a substantial fraction (30–40%) of the O3 entering leaves (at environmentally relevant concentrations) may be intercepted by apoplastic ASC in Vicia faba plants per se. Similar findings have been reported for Phaseolus vulgaris L. (Moldau et al., 1997, 1998), where ~50% of the incoming O3 was calculated to be detoxified by cell wall ASC. These findings suggest that the ASC pool located in the leaf apoplast may play a significant role as a first-line of defence against O3 in some species, a view supported by several independent lines of evidence: (i) the concentration of ASC in the cell wall is relatively high, ranging from 0.01 to 4.00 mM (Castillo and Greppin, 1988; Luwe et al., 1993; Polle et al., 1995; Kollist et al., 1996; Luwe, 1996; Ranieri et al., 1996); (ii) the biomolecular rate constant for the reaction of O3 with ASC (4.8x107 M-1 s-1 at pH 6.0–7.0; Kanofsky and Sima, 1995a), is higher than that for other regular constituents of the apoplast or components of the plasma membrane (Giamalva et al., 1985; Heath, 1988; Chameides, 1989; Kanofsky and Sima, 1995a; Mudway and Kelly, 1998); (iii) extracellular ASC content has been shown to be inversely correlated with O3 damage (Luwe et al. 1993; Barnes et al., 1999b; Kelly et al., 1995); (iv) manipulation of leaf ASC content (through feeding ASC, precursors or administering low/high light treatments) leads to predictable changes in O3 tolerance—increased levels of ASC affording enhanced protection (Freebairn, 1960; Freebairn and Taylor, 1960; Menser, 1964; Mächler et al., 1995; Zheng et al., 2000; Maddison and Barnes, unpublished results) and decreased levels enhancing O3 injury (Moldau et al., 1998); (v) O3 resistance (assessed in terms of visible injury) co-segregates with the capacity to synthesize ASC in a range of mutants of Arabidopsis thaliana L. (Conklin et al., 1996).
In the present study, model estimates indicated that at 75 nmol mol-1 O3 a significant fraction of the O3 flux (0.53 nmol O3 m-2 s-1) escaped interception through direct reaction with cell wall ASC and might therefore be expected to impinge on the plasmalemma. Yet, leaves developed no visible symptoms of O3 injury, and there were no significant changes in growth, root:shoot dry matter partitioning, intracellular ASC redox status or rates of CO2 assimilation, following 28 d exposure to the pollutant. This is an interesting observation that is not consistent with model estimates of JPLASMA and suggests that factors in addition to ASC most probably play a role in intercepting O3 in the leaf apoplast. This contention is supported by comparable data for hybrids of Populus deltoides (Ranieri et al., 1999), and for Triticum aestivum L. and Hordeum vugare L. (Kollist et al., 1996).
In conclusion, the findings of the present work indicate that ASC has an important role to play in the interception of O3 in the leaf cell walls of Vicia faba. However, the protection afforded is not complete, and the fate of the remaining O3 is unclear. It appears likely that additional apoplastic constituents may be as important as ASC in the detoxification of O3 in the leaf apoplast. The nature of these defences remain to be established.
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Acknowledgments |
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The authors sincerely thank Phil Green, Alan White and Keith Taylor for technical assistance. The work was financed through the Royal Society's East–West Postdoctoral Exchange Scheme with additional support to cover the day-to-day running costs of the fumigation facility provided by The Royal Society, Newcastle University Equipment Fund, The Swales Foundation, and the Overseas Development Agency. TL is indebted to the Department of Agricultural and Environmental Science (Newcastle University) for their financial support. The work was undertaken in JBs laboratory during his tenure of a Royal Society Research Fellowship.
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Notes |
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4 To whom correspondence should be addressed. Fax: +44 191 2225229. E-mail: J.D.Barnes{at}ncl.ac.uk
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References |
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